Abstract: Objective: The aim of the present study was to screen and to identify the cytotoxic compound from the leaves of Kigelia pinnata. Materials and Methods: Dry leaf powder of K. pinnata was extracted with chloroform and the extract was tested for anticancer cytotoxic activity by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide (MTT) assay in human colorectal carcinoma (HCT-116) cells. Crude extract was purified by silica gel column chromatography and the active fraction was identified by using spectroscopic techniques such as nuclear magnetic resonance, fourier transform-infrared spectroscopy and gas chromatography-mass spectrometry analysis. The identified compound was docked with cancer drug target protein, DNA topoisomerase-I using AutoDock4. Results: Crude extract showed significant anticancer cytotoxic activity with IC50 value of 80 μg mL1. The GC-MS chromatogram showed the presence of more than 15 different phytochemical compounds. The active fraction of the crude was identified as N-hexadecanoic acid with a molecular weight 256.42 Da and molecular formula C16H32O2. Molecular docking analysis showed that N-hexadecanoic acid has high affinity interaction with DNA topoisomerase-I with a free binding energy -6.71 kcal mol1. The N-hexadecanoic acid demonstrated significant IC50 value of 0.8 μg mL1 against HCT-116 cells. Conclusion: Based on the results of docking studies, it is proposed that the observed cytotoxic activity of N-hexadecanoic acid is due to its interaction with DNA topoisomerase-I and itcould be explored further for its anticancer cytotoxic potential with other cancer drug target proteins.